Our long-term goals are to characterize molecular mechanisms of virulence regulation in important bacterial pathogens and develop novel therapeutics against them. We will focus on iron-regulation of virulence factors in Corynebacterium diphtheriae and Mycobacterium tuberculosis, the etiologic agents of diphtheria and tuberculosis, respectively. Diphtheria, which re-emerged as a major epidemic disease in Russia and the Newly Independent States of the former Soviet Union during the 1990s, is a paradigm for pathogenesis of toxin-mediated bacterial diseases. Tuberculosis, which causes more deaths worldwide than any other bacterial infection, is a paradigm for pathogenesis of intracellular bacterial infections. Highly similar iron-activated regulatory proteins, called the diphtheria toxin repressor (DtxR) in C. diphtheriaee and the iron-dependent regulator (IdeR) in M. tuberculosis, control virulence in these very different pathogens. DtxR and IdeR are prototypes for a large family of metal-dependent global regulators found predominantly in Gram-positive and acid-fast bacteria. In Aim 1, we will identify iron-regulated genes and gene products in C. diphtheriaee by reporter transposon mutagenesis, proteomics and transcriptional arrays; characterize DtxR-dependent and DtxR-independent mechanisms of iron regulation; and study representative examples of these regulatory mechanisms at the molecular level. In Aim 2, we will characterize the siderophore-dependent iron-uptake pathway that interacts with DtxR to maintain iron homeostasis in C. diphtheriaee. We will determine the structure of the C. diphtheriaee siderophore called diphtheriabactin, compare it with the chemically distinct siderophore called corynebactin from C. glutamicum and B. subtilis, identify genes required for synthesis and export of diphtheriabactin, and characterize components of the Fe3+-diphtheriabactin uptake pathway both functionally and structurally. In Aim 3, we will determine the structures of wild-type and mutant forms of DtxR and IdeR that provide insights into their biological function; investigate hyperactive variants of these regulators to learn more about how they become activated and what structural features stabilize their fully active conformations; and use structure-based methods to design peptides and small molecules that will modulate IdeR activity, decrease virulence of M. tuberculosis, and have potential value as novel therapeutic agents for treatment of tuberculosis.